Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

Abstract

Gelsolin amyloidosis is an autosomal dominant incurable disease caused by a point mutation in the GSN gene (G654A/T), specifically affecting secreted plasma gelsolin. Incorrect folding of the mutant (D187N/Y) second gelsolin domain leads to a pathological proteolytic cascade. D187N/Y gelsolin is first cleaved by furin in the trans-Golgi network, generating a 68 kDa fragment (C68). Upon secretion, C68 is cleaved by MT1-MMP-like proteases in the extracellular matrix, releasing 8 kDa and 5 kDa amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. We developed nanobodies that recognize the C68 fragment, but not native wild type gelsolin, and used these as molecular chaperones to mitigate gelsolin amyloid buildup in a mouse model that recapitulates the proteolytic cascade. We identified gelsolin nanobodies that potently reduce C68 proteolysis by MT1-MMP in vitro. Converting these nanobodies into an albumin-binding format drastically increased their serum half-life in mice, rendering them suitable for intraperitoneal injection. A 12-week treatment schedule of heterozygote D187N gelsolin transgenic mice with recombinant bispecific gelsolin-albumin nanobody significantly decreased gelsolin buildup in the endomysium and concomitantly improved muscle contractile properties. These findings demonstrate that nanobodies may be of considerable value in the treatment of gelsolin amyloidosis and related diseases.

A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

“…The application of nanobodies as intracellular probes has already been demonstrated in numerous cases (10,11,13,16,17,63). In addition, Nb139 can be used as a stepping stone for the rational design of gain-of-function inhibitors.…”

A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study

“…It is therefore important, during therapy development, to test promising compounds over a long period of time and assess the effects at different time points during the course of the trial. During the Nb11 experiments, this had to be done by using different groups for every time point since the current methods of AGel analysis are end-stage [8,9]. Not only does this greatly augment the amount of mice needed, it also makes it impossible to evaluate the drug effect and amyloid build-up in a continuous manner.…”

“…This has far-reaching consequences in terms of molecular therapeutic avenues that should be followed when gelsolin has to be protected from cleavage by either of these proteases. As the second cleavage step occurs in the extracellular matrix, we reasoned that it might be possible to block this process by intraperitoneal administration of C68 chaperone nanobodies [8].…”

“…In a first approach, nanobodies, which partly protect C68 against MT1-MMP, were intraperitoneally injected in AGel mice [8]. In a second approach, a mouse model expressing Nb11 was developed [9].…”

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

“…The Nb not only positively affects transgenic mutant gelsolin proteostasis in skeletal muscle tissue but also attenuates the decrease in contraction speed of the extensor digitorum longus in an 8-min fatigue protocol [24]. Using adeno-associated virus as a vehicle, a bispecific nanobody was introduced in these mice that protects against both furin and MT1-MMP, yielding similar effects on muscle contraction speed [64,65]. Inhibiting the enzymatic activity of furin could be an alternative strategy, and noncompetitive furin-inhibiting nanobodies have been identified although they have not been tested for treatment of gelsolin amyloidosis [66].…”